Discovery c18 column

EHPr was analyzed by HPLC in order to identify their chemical composition.
Chromatographic analysis was performed according to [83 (link)]. Briefly, a Waters 2695 separation module system equipped with a Waters 996 photodiode array detector and Empower Pro software (Waters Corporation, USA) was used. Chemical separation was achieved using a Discovery C18 column (4.6 × 250 mm i.d., 5-μm particle size) (Sigma-Aldrich, Bellefonte, PA, USA). Two gradient elution methods were used. For both methods, the mobile phase consisted of a 0.5% trifluoroacetic acid aqueous solution (solvent A) and acetonitrile (solvent B). The gradient system of the first method was as follows: 0–1 min, 0% B; 2–3 min, 5% B; 4–20 min, 30% B; 21–23 min, 50% B; 24–25 min, 80% B; 26–27 100% B and 28–30 min, 0% B. The flow rate was maintained at 0.9 mL/min and the sample injection volume was 10 μL of sample diluted in methanol. 5-O-caffeoylquinic acid (chlorogenic acid), 4-O-caffeoylquinic acid (cryptochologenic acid), ferulic acid, quercetin3-O-glucoside and kaempferol-3-O-glucoside analytical standards were purchased from Sigma-Aldrich^®. Content of compounds in the extract was determined according to areas under the curve.

HPLC mobile phase was prepared by mixing 65:35:1 ratio of methanol, HPLC-grade water, and glacial acetic acid [40 ]. The diluent was prepared in a 100 mL volumetric flask by combining 1.50 g of calcium chloride dihydrate into 5.0 mL of HPLC-grade water. This mixture was agitated until calcium chloride dihydrate was completely dissolved. The resulting mixture was made to the final volume using methanol. The drug concentrations were determined using a Discovery C18 column (Sigma-Aldrich, Saint Louis, MO, USA) with 5 µm particle size, L × ID 150 mm × 4.6 mm [40 ]. Flow rate was set to isocratic 1.50 mL/min, and sample injection volume selected was 20 µL. Sample run time was set to 10 min with a column temperature of 30 °C. The expected analyte retention time (RT) for the drug peak was at approximately 4.0 min. Desoximetasone drug quantification was performed using a validated HPLC method on an Agilent 1100 series (Agilent Technologies, Santa Clara, CA, USA) coupled with UV detection (DAD) at a wavelength λmax 254 nm and HP ChemStation software v. 32.

The RG and HYFRG samples were extracted with 10-fold volume of methanol, filtered through a 0.45 μm membrane, and then analyzed by an Agilent 1200 series HPLC system (Agillent, Foster City, CA, USA) using Discovery C₁₈ column (250 × 4.6 mm, 5 μm, Sigma-Aldrich, MO, USA) under UV detector at 203 nm. The injection volume was 5 μL. The mobile phase consisted of acetonitrile (solvent A) and water (solvent B) applied by gradient at a flow rate of 1.6 mL/min. Gradient conditions were as follows: solvent A/solvent B (15/85, 20/80, 39/61, 48/52, 70/30, 90/10, 90/10, 15/85, and 15/85) with run times (0–5, 5–17, 17–57, 57–70, 70–80, 80–82, 82–92, 92–94, and 94–100 min), respectively.

The HPLC instrument used was Agilent 1100 series equipment (Agilent Technologies, Santa Clara, CA, USA) coupled with UV detection (DAD) and HP ChemStation software V. 32. For the analysis of desoximetasone, a mobile phase of 60% methanol and 40% water was pumped through a Discovery C18 column (Sigma-Aldrich, Saint Louis, MO, USA) with 5 µm particle size, L × ID 150 mm × 4.6 mm column. Injection volumes of 20 µL with a flow rate of 1.00 mL/min set to 30 °C with UV detection of 254 nm was used with the retention time of 4 min. The receptor media of the permeation study was utilized as diluent.